Identification of Ft proteins released in liquid culture and in macrophages. We have identified the major proteins released by Ft growing in liquid culture and demonstrated that several also are released within macrophages (25). Proteomic analysis of highly purified phagosomes. Our studies of Ft have yielded novel procedures for preparing highly purified Ft phagosomes and analyzing phagosome composition by Western immunoblotting and mass spectrometry (MS)-based proteomics. Transmission electron microscopy (TEM) demonstrates high phagosome purity with minimal contamination (Fig. i). Western immunoblotting of our purified phagosomes demonstrates that they have little or no endoplasmic reticulum or mitochondrial contamination. The same technique demonstrates that live LVS phagosomes have markedly reduced LAMP2 levels compared with those containing killed LVS, despite comparable amounts of the LVS marker protein, bacterioferritin. We have analyzed the host protein content of live and killed LVS phagosomes purified from THP-i macrophages by a MS based proteomic method, in collaboration with Dr. Loo and the RCE proteomic core facilities. Proteins we identified on live but not killed LVS phagosomes include the signaling and regulatory proteins, myotubularin, myotubularin-related protein-i (MTMRi), lamellipodin, ADP ribosylation factor like protein-3 (Arls), and syntaxin 11. Proteins only on killed LVS phagosome include Raby and the 16 kDa subunit of the vATPase. Proteins we identified on both live and killed phagosomes include CDi3, actin, and talin. Western immuno-blotting revealed that live (versus killed) LVS phagosomes have markedly reduced staining for flotillin-2 and vATPase (Fig. 2A). Copine-i, a phospho-lipid binding protein involved in signal transduction, on the other hand, is present at comparable levels on live and killed LVS phagosomes (Fig. 2A). Western immunoblotting has confirmed our proteomic finding of MTMRi on live but not killed LVS phagosomes (Fig. 2B). Conversely, Western immunoblotting demonstrates enrichment of Neiman Pick Protein C-i (NPCl), TPCi-e, cathepsin D, and galectin-i on killed LVS phagosomes versus their absence or scarcity on live LVS phagosomes (Fig. 2B).